A significant problem with many of the currently available molecular biology techniques is their reliance upon naturally occurring convenient restriction sites. Modifications of the polymerase chain reaction (PCR) and other similar amplification techniques have been developed in an attempt to overcome this problem. In the absence of naturally occurring convenient restriction sites, it is possible to introduce restriction sites into the sequence of interest by using primers and PCR. However, this technique results in the presence of extraneous polynucleotides in the amplification products even after restriction digestion. Such extraneous polynucleotides can be problematic. For example, the introduction of unwanted nucleotides often imposes design limitations on the cloned product which may interfere with the structure and function of the desired gene products.
One method of joining DNA without introducing extraneous bases or relying on the presence of restriction sites is splice overlap extension (SOE). Yon et al., 1989, Nucl. Acids Res. 17:4895. Horton et al., 1989, Gene 77:61-68. This method is based on the hybridization of homologous 3' single-stranded overhangs to prime synthesis of DNA using each complementary strand as template. Although this technique can join fragments without introducing extraneous nucleotides (in other words, seamlessly), it does not permit the easy insertion of a DNA segment into a specific location when seamless junctions at both ends of the segment are required. Nor does this technique function to join fragments with a vector. Ligation with a vector must be subsequently performed by incorporating restriction sites onto the termini of the final SOE fragment. Finally, this technique is particularly awkward when trying to exchange polynucleotides encoding various domains or mutation sites between genetic constructs encoding related proteins.
Another commonly used genetic manipulation technique is immobilized amplification, e.g., immobilized PCR. In techniques involving immobilized PCR, i.e., bound PCR, polynucleotide amplification products are immobilized on a solid phase support. Immobilization is typically accomplished through the use of streptavidin (or avidin) and biotinylated polynucleotides, antibody-hapten binding interactions, or through the covalent attachment of nucleic acids to solid supports. A serious limitation, however, of such conventional immobilization techniques is that the amplification products cannot be conveniently unbound from the solid phase support for use in subsequent manipulations, e.g., sequencing of the amplification products.
An additional problem with conventional techniques, particularly the manipulation of amplification reaction products, is that cleavage at certain restriction sites must be avoided in order to obtain desired polynucleotides. Presently, however, this can only be accomplished by cumbersome techniques such as partial digestions and methylase protection.
Accordingly, in view of the foregoing limitations of current recombinant DNA technology, it is of interest to provide improved techniques for conveniently manipulating polynucleotides without having to rely on naturally occurring convenient restriction sites. It is also of interest to provide methods of synthesizing polynucleotides in which some or all of the nucleotides introduced through synthesis primers may be conveniently removed from the final synthesis product. Additionally, it is of interest to provide improved methods of manipulating polynucleotide synthesis products by restriction enzymes which overcome the problems of cleavage at internal sites within the synthesis products. Further, it is of interest to provide an improved method of releasing amplification products that are bound to a solid phase support. The present invention meets these needs.